scholarly journals Methylthioadenosine toxicity and metabolism to methionine in mammalian cells

1988 ◽  
Vol 255 (1) ◽  
pp. 145-152 ◽  
Author(s):  
L Christa ◽  
J Kersual ◽  
J Augé ◽  
J L Pérignon
Keyword(s):  
Total Synthesis ◽  
Toxic Effect ◽  
Mammalian Cells ◽  
Resting Cells ◽  
Free Medium ◽  
Raji Cells ◽  
Polyamine Synthesis ◽  
Ccl39 Cells ◽  
Growth Experiments ◽  
Metabolic Cycle

5′-Deoxy-5′-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5′-deoxy-5′-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.

Preparation of individual human diploid fibroblasts and study of ion transport

1985 ◽  
Vol 248 (1) ◽  
pp. C154-C164 ◽  
Author(s):  
E. H. Abraham ◽  
J. L. Breslow ◽  
J. Epstein ◽  
P. Chang-Sing ◽  
C. Lechene
Keyword(s):  
Mammalian Cells ◽  
Effective Permeability ◽  
Chloride Content ◽  
Cellular Transport ◽  
Free Medium ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Cellular Volume ◽  
Intracellular Content ◽  
Individual Human

A method for analyzing individual mammalian cells with electron probe microanalysis has been developed using human diploid fibroblasts. Cells were grown on the same support that is used for experimental manipulations and analysis. Steady-state cation and anion concentrations and kinetic processes during experimental perturbations could be measured on populations of less than 1,000 cells. Human diploid fibroblasts in normal tissue culture medium had the following intracellular concentrations (in mM): K, 168; Na, 25.0; Cl, 51.2; P, 84.1; S, 16.5; Ca, 6.04; and Mg, 10.0. The ratios of K to Na were equivalent when measured in the nuclear or cytoplasmic area of the cells. Serum in the incubation medium was found to increase the cellular effective permeability to Na by a factor of 2.5, while leaving the effective permeability to K unchanged. When returned to control medium after 7 h of incubation in K-free medium, the cells recovered normal K/Na in less than 1 h. In some experiments the coupling ratio of the ouabain-inhibitable cellular transport of Na to K was 3:2 and the ratio of Cl to K was 1:2. The sum of intracellular content (Na + K) (an estimate of cellular volume) did not change when the cells were placed in K-free medium and increased by less than 30% after ouabain treatment. After 5-7 h of ouabain treatment or of incubation in K-free medium, long after the intracellular K had been replaced by Na, the cellular chloride content had not changed significantly.

scholarly journals Polyamines and Their Role in Virus Infection

2017 ◽  
Vol 81 (4) ◽  
Author(s):  
Bryan C. Mounce ◽  
Michelle E. Olsen ◽  
Marco Vignuzzi ◽  
John H. Connor
Keyword(s):  
Protein Synthesis ◽  
Nucleic Acid ◽  
Mammalian Cells ◽  
Cellular Proliferation ◽  
Polyamine Synthesis ◽  
Host Interactions ◽  
Dna And Rna ◽  
Rna Polymerization ◽  
Nucleic Acid Conformation

SUMMARY Polyamines are small, abundant, aliphatic molecules present in all mammalian cells. Within the context of the cell, they play a myriad of roles, from modulating nucleic acid conformation to promoting cellular proliferation and signaling. In addition, polyamines have emerged as important molecules in virus-host interactions. Many viruses have been shown to require polyamines for one or more aspects of their replication cycle, including DNA and RNA polymerization, nucleic acid packaging, and protein synthesis. Understanding the role of polyamines has become easier with the application of small-molecule inhibitors of polyamine synthesis and the use of interferon-induced regulators of polyamines. Here we review the diverse mechanisms in which viruses require polyamines and investigate blocking polyamine synthesis as a potential broad-spectrum antiviral approach.

scholarly journals Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochromec

2002 ◽  
Vol 282 (6) ◽  
pp. C1290-C1297 ◽  
Author(s):  
Qing Yuan ◽  
Ramesh M. Ray ◽  
Leonard R. Johnson
Keyword(s):  
Cytochrome C ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Intestinal Epithelial ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Polyamine Synthesis ◽  
Antiapoptotic Proteins ◽  
Induced Apoptosis ◽  
Rate Limiting

C1297, 2002; 10.1152/ajpcell.00351.2001.We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome cfrom mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with α-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-xL and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.

Intracellular free Na+ in resting and activated A7r5 vascular smooth muscle cells

1993 ◽  
Vol 264 (6) ◽  
pp. C1513-C1524 ◽  
Author(s):  
M. L. Borin ◽  
W. F. Goldman ◽  
M. P. Blaustein
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cell Activation ◽  
Prolonged Exposure ◽  
Muscle Cells ◽  
Resting Cells ◽  
Free Medium ◽  
Na Pump

Regulation of intracellular Na+ ([Na+]i) in cultured vascular smooth muscle cells (A7r5 line) was studied with Na(+)-sensitive fluorescent dye sodium-binding benzofuran isophthalate. Digital imaging microscopy was used to study single-cell fluorescence. Na+ was distributed uniformly in cytoplasm and nucleus; mean Na+ concentration in resting cells was 4.4 +/- 0.3 mM in cytoplasmic areas ([Na+]cyt) and 4.5 +/- 0.4 mM in nuclear areas ([Na+]n). Na+ pump inhibition and cell activation evoked uniform changes in [Na+]cyt and [Na+]n. Inhibition of Na+ pump with 1 mM ouabain or K(+)-free medium caused a rise in [Na+]cyt; in the latter case, [Na+]cyt fell rapidly when external K+ was later restored. Exposure to Ca(2+)-free medium also caused [Na+]cyt to rise; this effect was augmented by Na+ pump inhibition and was reversed by 10(-5) M verapamil or nitrendipine or by restoration of external Ca2+. The implication is that this Na+ entry in absence of external Ca2+ is mediated by Ca2+ channels. Activation by 10(-9) M arginine vasopressin (AVP) and 10(-6) M serotonin (5-HT) caused [Na+]cyt to increase, but response to 5-HT was small (0.6 mM on average) and transient, whereas response to AVP was larger (2.4 mM on average) and was maintained as long as AVP was present (to 20 min). AVP and, to a much smaller extent, 5-HT stimulated Na+ influx; this could be detected when Na+ pump was inhibited by ouabain. Both AVP and 5-HT activated the Na+ pump, as detected by ouabain-sensitive decrease in [Na+]cyt when Na+ influx was inhibited. Agonist-evoked increases in [Na+]cyt were dependent on a rise in cytosolic Ca2+ concentration ([Ca2+]cyt); these [Na+]cyt responses were abolished by prolonged exposure to Ca(2+)-free media, when cytoplasmic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or when Ca2+ mobilization was blocked with thapsigargin. Raising [Ca2+]cyt with 40 mM K+ or with thapsigargin did not increases in [Na+]cyt. We conclude that 1) AVP- and 5-HT-evoked increases in [Na+]cyt are agonist specific and depend on the balance between stimulated Na+ influx and efflux; 2) AVP and 5-HT activate the Na+ pump; this is, at least in part, independent of agonist-induced rise in [Na+]cyt; and 3) a rise in [Ca2+]cyt is necessary but not sufficient to trigger agonist-evoked rise in [Na+]i.

scholarly journals DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells

2009 ◽  
Vol 38 (1) ◽  
pp. 339-352 ◽  
Author(s):  
Sivaprasad Attaluri ◽  
Radha R. Bonala ◽  
In-Young Yang ◽  
Mark A. Lukin ◽  
Yujing Wen ◽  
...  
Keyword(s):  
Total Synthesis ◽  
Dna Adducts ◽  
Mammalian Cells ◽  
Aristolochic Acid ◽  
Site Specific

scholarly journals THE POPULATION-DEPENDENT REQUIREMENT BY CULTURED MAMMALIAN CELLS FOR METABOLITES WHICH THEY CAN SYNTHESIZE

1962 ◽  
Vol 116 (1) ◽  
pp. 29-43 ◽  
Author(s):  
Harry Eagle ◽  
Karl Piez
Keyword(s):  
Population Density ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Free Medium ◽  
Experimental Conditions ◽  
Cellular Survival ◽  
Sustained Growth ◽  
Cell Line Hela ◽  
Specific Organ ◽  
Cell Densities

At least seven compounds synthesized by cultured cells in amounts which should suffice for sustained growth have nevertheless proved under certain conditions necessary for survival (asparagine, cystine, glutamine, homocystine inositol, pyruvate, serine). In every instance so far examined, that requirement has been population-dependent, disappearing at cell densities sufficiently large to bring the concentration in the medium and in the cellular pool to metabolically effective levels before the cells died of the specific deficiency. At population densities of less than 100 cells per ml, serine was required by all cultured cells so far studied. With more exigent strains, such as the RT6 strain of rabbit fibroblast and the P388 mouse leukemia, the serine requirement disappeared only at populations in excess of 50,000 and 150,000 cells per ml, respectively. The requirement for pyruvate by the latter cell as an alternative to serine also disappeared at that population density. In a cystine-free medium there were population-dependent requirements for cystine, homocystine, or serine, depending on the experimental conditions. With methionine and glucose as cystine precursors, the critical population density permitting cellular survival and growth was in excess of 200,000 cells/ml. The provision of homocystine as an intermediate reduced the critical population density to 10,000 to 60,000 cells/ml; with the further provision of serine, the critical cell concentration permitting growth was reduced to 50 to 500 cells/ml. Cells adapted to glutamic acid, and capable of utilizing it as a substitute for glutamine, nevertheless required exogeneous glutamine at cellular densities of less than 50,000 cells per ml. In some experiments, the provision of asparagine reduced the critical population density to 10,000 cells/ml, presumably because of its glutamine-sparing action. Inositol is required by most cell lines, despite their demonstrated capacity to synthesize it from glucose. With at least one cell line (HeLa), sustained growth was occasionally achieved in an inositol-free medium if the population density was maintained in excess of 240,000 cells/ml. The possible implications of these findings with respect to the loss of specific organ functions in dispersed cell culture are discussed.

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